Summary of Work: Hereditary mutations of the BRCA1 and BRCA2 genes confer a profound predisposition to breast and ovarian cancer in women. Our group has used gene targeting in an attempt to create experimental mouse models for Brca defects. Several low-level gamma radiation studies (0.3 Gy) are in progress with hemizygous Brca1-deficient, Brca2-deficient, Brca1-deficient x Brca2-deficient F1, and mutant P53 x Brca2-deficient F1 mice carrying disruptions of exon 10 and/or 11. Preliminary results from these experiments suggest that haploinsufficiency for the Brca genes does not strongly predispose mice to neoplastic development relative to their wild type littermates. In contrast, examination of mammary gland whole mounts from Brca1- and Brca2-deficient mice at 6 months of age revealed a significant inhibition of ductal morphogenesis as compared to their respective normal littermates. Although the mammary ducts appear to elongate normally, lateral and side branching and alveolar bud formation are reduced in haploinsufficient Brca mice relative to wild type littermates. In addition, we have recently completed a survey of several inbred mouse strains for susceptibility to mammary tumor formation. This study demonstrated that BALB/cJ and SWR/J mice are considerably more susceptible to low dose radiation-induced mammary carcinogenesis (~20 & 40% incidence at 2 years of age, respectively) compared to other commonly used inbred strains (C57BL/6, C3H/He, FVB/N, and 129/SvEv). We have backcrossed our Brca2 exon 10/11 mutation onto the susceptible BALB/cJ background and used them in mammary transplantation studies with gamma radiation doses ranging between 0.3 and 6 Gy. Transplantation recipient mice are being monitored currently for development of dysplastic mammary lesions and mammary tumors. Efforts to develop Brca mouse models have been inhibited by the early embryonic lethality of homozygous mutant mice. We have overcome this difficulty during the past year through use of the Cre-loxP system. Gene targeting was used to generate mice carrying a pair of loxP sites flanking exon 27 of the Brca2 gene. This carboxy terminal domain of the Brca2 gene product interacts directly with the Rad51 DNA repair protein and appears to be critical for maintaining genome stability in response to various types of DNA damage. Intercrosses hve been performed between our floxed Brca2 mice and two different strains of MMTV-Cre transgenic mice that express the Cre recombinase under the control of the Mouse Mammary Tumor Virus promoter. Offspring from both crosses are under careful observation for neoplastic development in mammary tissue as well as other organs. Female pups from the MMTV-Cre Strain D cross should delete both floxed Brca2 exon 27 alleles homozygously in most mammary epithelial cells during puberty. We have confirmed the specificity of these Cre-mediated deletions in a preliminary analysis of MMTV-Cre Strain D x ROSA26LacZ-Cre reporter F1 mice. In contrast, floxed Brca2 alleles that pass through female MMTV-Cre Strain A mice are completely deleted in the germline of the resulting offspring. Intercrosses of these Brca2 exon 27-deleted offspring have demonstrated that homozygous mutant mice are viable and they lack apparent phenotypes up to at least seven weeks of age. Test matings are in progress to determine if these homozygous Brca2-mutant mice are fertile. Finally, we have deleted Brca2 exon 27 in our floxed embryonic stem cells by transient transfection with a Cre expression plasmid and used these cells to generate hemizygous mutant Brca2 mice. Intercrosses of these mice are in progress and homozygous mutant offspring will be followed to study neoplastic development in mammary gland and other tissues which results from homozygous disruption of the Brca2 carboxy terminal domain. Mice that are homozygous for floxed or deleted exon 27 alleles of the Brca2 gene will provide an invaluable source of primary cells and cell lines for biochemical studies of Brca2 function in vitro. - Breast cancer, Brca1, Brca2, Brca2-deficient mice, Cre-loxP, radiation